The primary objective of this project is to examine the mechanisms that regulate the intracellular trafficking of membrane receptors, and particularly membrane immunoglobulins (mIg's) in their role in internalizing antigen and efficiently targeting it for processing and presentation to T cells. 1. Intracellular trafficking of mIg and antigen in B cell mIg transfectants: Murine B cell lines transfected with mutant human mIgM's with phosphorylcholine-specific V regions have identified a transmembrane tyrosine (TYR587) critical for efficient antigen processing and presentation. The intracellular fate of these mIgM mutants and associated antigen will be analyzed by subcellular fractionation, using a novel ELISA technique, and by immunoelectron microscopy. These intracellular pathways will also be compared to those taken by antigen endocytosed via i) pinocytosis and ii) Fc-receptors. 2. Identification of transmembrane residues and associated molecules that direct intracellular targeting of membrane receptors: The role of specific transmembrane residues in intracellular targeting will be directly addressed by site-directed mutation and transfection of additional mIgM constructs or human HLA-A2 (class I MHC molecule) constructs into murine B cell lines; intracellular trafficking will then be analyzed by subcellular fractionation and immunoelectron microscopy. Using the mIgM and HLA mutant clones, molecules specifically associated with the mutated and uniquely targeted receptors (presumably responsible for intracellular targeting) will be identified. Kinetic analyses will indicate whether associated proteins actively "chaperon" receptors to selected organelles, or whether they are static and only "moor" receptors upon entering specific intracellular sites. 3. Detection of processed peptides in antigen presenting cells and characterization of the processing compartment: A novel assay has been developed to detect functional stimulatory MHC II-peptide complexes within subcellular fractions. This technique will be used to identify the compartment where internalized antigen is processed ("endosome"); isolated endosomes will then be biochemically characterized. Thus, this project will utilize novel assays and a powerful set of transfected B cell clones to analyze questions relevant not only to antigen processing, but also to more general questions related to intracellular receptor trafficking.